Actomyosin contractility is a major engine of preimplantation morphogenesis, which starts at the 8-cell stage during mouse embryonic development. Contractility becomes first visible with the appearance of periodic cortical waves of contraction (PeCoWaCo), which travel around blastomeres in an oscillatory fashion. How contractility of the mouse embryo becomes active remains unknown. We have taken advantage of PeCoWaCo to study the awakening of contractility during preimplantation development. We find that PeCoWaCo become detectable in most embryos only after the second cleavage and gradually increase their oscillation frequency with each successive cleavage. To test the influence of cell size reduction during cleavage divisions, we use cell fusion and fragmentation to manipulate cell size across a 20- to 60-μm range. We find that the stepwise reduction in cell size caused by cleavage divisions does not explain the presence of PeCoWaCo or their accelerating rhythm. Instead, we discover that blastomeres gradually decrease their surface tensions until the 8-cell stage and that artificially softening cells enhances PeCoWaCo prematurely. We further identify the programmed down-regulation of the formin Fmnl3 as a required event to soften the cortex and expose PeCoWaCo. Therefore, during cleavage stages, cortical softening, mediated by Fmnl3 down-regulation, awakens zygotic contractility before preimplantation morphogenesis. During preimplantation morphogenesis, the mouse embryo relies on forces generated by the actomyosin cytoskeleton. This study uncovers how periodic actomyosin contractions increase in frequency during cleavage stages as blastomeres soften with each cleavage division. During embryonic development, the shape of animal cells and tissues largely relies on the contractility of the actomyosin cortex –3. The aMyh9) rather than IIB (encoded by Myh10) [During mouse preimplantation development, PeCoWaCo become visible before compaction , the firy Myh10) . Dissociy Myh10) . Interesy Myh10) . Similary Myh10) . This ray Myh10) ,35. Thisy Myh10) ,42.In this study, we investigate how the contractility of the cleavage stages emerges before initiating blastocyst morphogenesis. We take advantage of the slow development of the mouse embryo to study thousands of pulsed contractions and of the robustness of the mouse embryo to size manipulation to explore the geometrical regulation of PeCoWaCo. We discover that the initiation, maintenance, or oscillatory properties of PeCoWaCo do not depend on cell size. Instead, we discover a gradual softening of blastomeres with each successive cleavage, which exposes PeCoWaCo. This softening results in part from the reorganization of the actin cortex due to the down-regulation of the formin Fmnl3 during the first cleavage stages. Together, this study reveals how preimplantation contractility is robust to the geometrical changes of the cleavage stages during which the zygotic contractility awakens.PeCoWaCo have been observed at the 8-, 16-cell, and blastocyst stages. To know when PeCoWaCo first appear, we imaged embryos during the cleavage stages and performed Particle Image Velocimetry (PIV) and Fourier analyses . We noteFirst, to test whether the initiation of PeCoWaCo in most 4-cell stage embryos depends on the transition from the 2- to 4-cell stage blastomere size, we prevented cytokinesis. Using transient exposure to Vx-680 to inhibit the activity of Aurora kinases triggering chromosome separation, we specifically blocked the 2- to 4-cell stage cytokinesis without compromising the next cleavage to the 8-cell stage . This caThen, we tested whether PeCoWaCo could be triggered prematurely by artificially reducing 2-cell stage blastomeres to the size of a 4-cell stage blastomere. To reduce cell size, we treated dissociated 2-cell stage blastomeres with the actin cytoskeleton inhibitor Cytochalasin D before deforming them repeatedly into a narrow pipette . By adapThe transition from 2- to 4-cell stage blastomere size is neither required nor sufficient to initiate PeCoWaCo. Nevertheless, the decrease in period of PeCoWaCo remarkably scales with the stepwise decrease in blastomere size . Given aFusion of cells causes blastomeres to contain multiple nuclei, while cell fragmentation creates enucleated fragments. Interestingly, enucleated fragments continued oscillating with the same period and showing identical propagation velocities as the nucleus-containing fragments . These mTogether, using fusion and fragmentation of cells, we find that PeCoWaCo oscillation properties are robust to a large range of size perturbations. Therefore, other mechanisms must be at play to regulate periodic contractions during preimplantation development.Despite the apparent relationship between cell size and PeCoWaCo during preimplantation development, our experimental manipulations of cell size reveal that PeCoWaCo are not influenced by cell size. PeCoWaCo result from the activity of the actomyosin cortex, which could become stronger during cleavage stages and make PeCoWaCo more prominent as previously observed during the 16-cell stage . Since aTo investigate the changes responsible for blastomere softening during cleavage stages, we first looked into changes in cortical organization. Using super resolution microscopy on phalloidin-stained embryos, we measured the thickness of the actomyosin cortex, which has been reported to change with surface tension . Using lTo investigate the molecular changes responsible for blastomere softening during cleavage stages, we took advantage of available single-cell RNA sequencing and proteomic data . We noteTogether, these experiments using the pulsatile nature of cell contractility reveal the unsuspected maturation of the cortex of blastomeres during the cleavage stages of mouse embryonic development.During cleavage stages, blastomeres halve their size with successive divisions. Besides the increased number of blastomeres, there is no change in the architecture of the mouse embryo until the 8-cell stage with compaction. We find that this impression of stillness is only true on a timescale of hours since, on the timescale of seconds, blastomeres display signs of actomyosin contractility. During the first 2 days after fertilization, contractility seems to mature by displaying more frequent and visible pulses. We further find that pulsed contractions do not rely on the successive reductions in cell size but rather on the gradual decrease in surface tension of the blastomeres. Therefore, during cleavage stages, cortical softening awakens zygotic contractility before preimplantation morphogenesis.Previous studies on the cytoskeleton of the early mouse embryo revealed that both the microtubule and intermediate filament networks mature during cleavage stages. Keratin intermediate filaments appear at the onset of blastocyst morphogenesis  and becoThe maturation of zygotic contractility may be influenced by the activation of the zygotic genome occurring partly at the late zygote stage and mainly during the 2-cell stage ,54. ReceThe effect of mechanical constraints on pulsed contractions is reminiscent of recent reports in fly embryos in which Fmnl-mediated densification of a persisting actin cytoskeleton dampens pulsed contractions . In addiTogether, our study uncovers the maturation of the actomyosin cortex, which softens and speeds up the rhythm of contractions during the cleavage stages of the mouse embryo. Interestingly, zebrafish embryos also soften during their cleavage stages, enabling doming, the first morphogenetic movement in zebrafish . It willAll animal work is performed in the animal facility at the Institut Curie, with permission by the institutional veterinarian overseeing the operation (APAFIS #11054–2017082914226001). The animal facilities are operated according to international animal welfare rules.Embryos are isolated from superovulated female mice mated with male mice. Superovulation of female mice is induced by intraperitoneal injection of 5 international units (IU) pregnant mare serum gonadotropin , SYNCRO-PART), followed by intraperitoneal injection of 5 IU human chorionic gonadotropin  44 to 48 hours later. Embryos are recovered at E0.5 by dissecting in 37°C FHM  from the oviduct the ampula, from which embryos are cleared with a brief (5 to 10 seconds) exposure to 37°C hyaluronidase .Embryos are recovered at E1.5 or E2.5 by flushing oviducts from plugged females with 37°C FHM using a modified syringe .Embryos are handled using an aspirator tube  equipped with a glass pipette pulled from glass micropipettes .2 at 37°C.Embryos are placed in KSOM  or FHM supplemented with 0.1% BSA  in 10 μL droplets covered in mineral oil  unless stated otherwise. Embryos are cultured in an incubator with a humidified atmosphere supplemented with 5% COTo remove the zona pellucida (ZP), embryos are incubated for 45 to 60 seconds in pronase .For imaging, embryos are placed in 5- or 10-cm glass bottom dishes .Only embryos surviving the experiments were analyzed. Survival is assessed by continuation of cell division as normal when embryos are placed in optimal culture conditions.tm4Luo) is used [Mice are used from 5 weeks old on. (C57BL/6xC3H) F1 hybrid strain is used for wild-type (WT). To visualize plasma membranes, mTmG (Gt(ROSA)26Sor is used .ZP-free 2-cell or 4-cell stages embryos are aspirated multiple times  through a smoothened glass pipette  until dissociation of cells.2+ free KSOM [For 8- and 16-cell stage embryos, they are placed into EDTA containing Caree KSOM  for 8 toVx-680  50 mM DMSO stock was diluted to 2.5 μM in KSOM. To prevent mitosis, 2-cell stage embryos are cultured in 2.5 μM Vx-680 for 3 hours shortly prior to the second cleavage and then washed in KSOM. Subsequent divisions were observed in 12/20 of the cases.Cytochalasin D  10 mM DMSO stock is diluted to 10 μM in KSOM. To fragment cells, isolated 2- or 16-cell stage blastomeres were treated with Cytochalasin D for 20 minutes before being gently aspirated into a smoothened glass pipette of diameter about 30 or 5 to 10 μm, respectively . MoreoveGenomONE-CF FZ SeV-E cell fusion kit  is used to fuse blastomeres . HVJ envLatrunculin A  10 mM DMSO stock is diluted to 100 nM in KSOM. To soften cells, 2-cell stage embryos are imaged in medium containing Latrunculin A covered with mineral oil for 2 hours.Fmnl3, we performed total RNA extraction from a pool of 50 zygotes using the PicoPure RNA Isolation Kit . DNase treatment is performed during the extraction, using RNase-Free DNase Set . Subsequently, a cDNA library is synthesized with oligo(dT)  using the Super-Script III Reverse Transcriptase kit  on all the extracted RNA, according to manufacturer’s instructions. As a final step, a fragment of 3,084 bp corresponding to Fmnl3 isoform 202 (MGI:109569) is specifically isolated from the cDNA library, by PCR amplification with forward (fw) and reverse (rv) primers GCATGGACGAGCTGTACAAGGGCAACCTGGAGAGCACCGA and TAGTTCTAGACCGGATCCGGCTAACAGTTTGACTCGTCATG, respectively.To isolate cDNA of GFP reporter gene, and Formin like 3 (Fmnl3) cDNAs, are initially generated by PCR amplification. During this step, overlapping ends are incorporated into each fragment.To generate the GFP-Fmnl3 plasmid construct for in vitro mRNA synthesis, the Gibson Assembly cloning method was used. Three linear DNA fragments, corresponding to pCS2+ backbone, Forward and reverse primers to obtain a 4,087-bp fragment of the pCS2+ backbone: TGACGAGTCAAACTGTTAGCCGGATCCGGTCTAGAACTATAGTGAGTCGT and AGTGAGTCGTATTACCGGATCCGGTCTATAGTGTCACCTAAATC.GFP: CGGTAATACGACTCACTATAGGCCGGATCCGGATGGTGAGCAAGGGCGAGGA and TCGGTGCTCTCCAGGTTGCCCTTGTACAGCTCGTCCATGC.Forward and reverse primers to obtain a 717-bp fragment encoding Following DNA purification, the assembly of the final construct is achieved by incubating the 3 fragments in the Gibson Assembly Master Mix , according to the manufacturer’s instruction.Following the linearization of the pCS2-GFP-Fmnl3 plasmid using Hind III, GFP-Fmnl3 mRNA is transcribed using the mMESSAGE mMACHINE SP6 Kit  according to manufacturer’s instructions and resuspended in Rnase-free water.GFP mRNA is generated by in vitro transcription of a GFP linear DNA fragment of approximately 750 bp obtained by PCR amplification from the pCS2-GFP-Fmnl3 plasmid, with fw primer ATTTAGGTGACACTATAGAGCC and rv primer CTACTTGTACAGCTCGTCCAT.Glass capillaries  glass capillaries with 780-μm inner diameter) are pulled using a needle puller and micro forged to forge a holding pipette and an injection needle. The resulting injection needles are filled with mRNA solution diluted to 1 μg/μL in injection buffer . The filled needle is positioned on a micromanipulator (Narishige MMO-4) and connected to a positive pressure pump (Eppendorf FemtoJet 4i). Embryos are placed in FHM drops covered with mineral oil under Leica  TL Led microscope. Two-cell stage embryos were injected while holding with holding pipette connected to a Micropump CellTram Oil.Rp). At steady state, the surface tension γ of the cell is calculated from the Young–Laplace’s law applied between the cell and the micropipette: γ = Pc / 2 (1/Rp—1/Rc), where Pc is the critical pressure used to deform the cell of radius of curvature Rc.As described previously ,64, a miEight-cell stage embryos are measured before compaction , during which surface tension would increase .Fragmented cells and their control cells are measured 10 to 15 hours after fragmentation. At that point, enucleated fragments are mostly irregular in shape and cannot be measured.Measurements of individual blastomeres from the same embryo are averaged and plotted as such.Embryos are fixed in 2% PFA  for 10 minutes at 37°C, washed in PBS, and permeabilized in 0.01% Triton X-100  in PBS (PBT) at room temperature before being placed in blocking solution (PBT with 3% BSA) at 4°C for 2 to 4 hours. Primary antibodies  are applFor live imaging, embryos are placed in 5-cm glass bottom dishes (MatTek) under a CellDiscoverer 7  equipped with a 20×/0.95 objective and an ORCA-Flash 4.0 camera  or a 506 axiovert (Zeiss) camera.2 at 37°C.Using the experiment designer tool of ZEN (Zeiss), we set up nested time-lapses in which all embryos are imaged every 3 to 5 hours for approximately 10 minutes with an image taken every 5 seconds at 2 focal planes positioned 10 μm apart. Embryos are kept in a humidified atmosphere supplied with 5% CO2.mTmG embryos are imaged at the 16-cell stage using an inverted Zeiss Observer Z1 microscope with a CSU-X1 spinning disc unit . Excitation is achieved using a 561 nm laser through a 63×/1.2 C Apo Korr water immersion objective. Emission is collected through 595/50 band-pass filters onto an ORCA-Flash 4.0 camera . The microscope is equipped with an incubation chamber to keep the sample at 37°C and supply the atmosphere with 5% CO2.Surface tension measurements are performed on a Leica DMI6000 B inverted microscope equipped with a 40×/0.8 DRY HC PL APO Ph2 (11506383) objective and Retina R3 camera and 0.7× lens in front of the camera. The microscope is equipped with an incubation chamber to keep the sample at 37°C and supply the atmosphere with 5% COStained embryos are imaged on a Zeis LSM900 Inverted Laser Scanning Confocal Microscope with Airyscan detector. Excitation is achieved using a 488-nm laser line through a 63×/1.4 OIL DICII PL APO objective. Emission is collected through a 525/50 band-pass filter onto an airyscan photomultiplier (PMT) allowing to increase the resolution up to a factor 1.7.Manual shape measurements. Fiji [ts. Fiji  is used PIV analysis. To detect PeCoWaCo in phase contrast images of embryos, we use PIV analysis followed by a Fourier analysis.As previously ,40, PIVlThe x- and y-velocities of individual vectors from PIV analysis are used for Fourier analysis. A Fourier transform of the vector velocities over time is performed using MATLAB’s fast Fourier transform function. The resulting Fourier transforms are squared to obtain individual power spectra. Squared Fourier transforms in the x and y directions of all vectors are averaged for individual embryos resulting in mean power spectra of individual embryos.Spectra of individual embryos are checked for the presence of a distinct amplitude peak to extract the oscillation period. The peak value between 50 seconds and 200 seconds was taken as the amplitude, as this oscillation period range is detectable by our imaging method. An embryo is considered as oscillating when the amplitude peaks 1.777 times above background . This threshold value was determined using CutOffFinder  S1 Fig)S1 Fig) tTwo-cell, 4-cell, and 8-cell stages are considered early during the first half of the corresponding stage and late during the second half.Since PeCoWaCo halt during mitosis , time laLocal curvature analysis. To measure PeCoWaCo period, amplitude, and velocity, we analyze the associated changes in surface curvature and perform Fourier analysis. Importantly, since we can only extract these parameters from oscillating blastomeres and embryos, data shown in WizardofOz, found under the Mtrack repository) for computing the local curvature information using the start, center and end point of a 10-μm strip on the cell surface to fit a circle. The strip is then moved by 1 pixel along the segmented cell, and a new circle is fitted. This process is repeated till all the points of the cell are covered. The radius of curvature of the 10-μm strip boundaries are averaged. Kymograph of local curvature values around the perimeter over time is produced by plotting the perimeter of the strip over time.To obtain the local curvature of isolated blastomeres and embryos, we developed an approach similar to that of ,36,70. FCurvature kymographs obtained from local curvature tracking are then exported into a custom made Python script for 2D Fast Fourier Transform analysis.Spectra of individual cells are checked for the presence of a distinct amplitude peak to extract the oscillation period. The peak value between 50 seconds and 200 seconds was taken as the amplitude, as this oscillation period range is detectable by our imaging method.To measure the wave velocity, a line is manually fitted on the curvature kymograph using Fiji.Cortex thickness measurement. Super resolution images obtained using airyscan microscopy are used to measure cortex thickness. The full width at half maximum of cortical intensity profiles were used to assess cortical thickness by using CortexThicknessAnalysis tool [https://github.com/PaluchLabUCL/CortexThicknessAnalysis.sis tool  availablt test, and chi-squared p-values are calculated using Excel (Microsoft) or R . Statistical significance is considered when p < 10−2.Data are plotted using Excel  and R-based SuperPlotsOfData tool . Mean, sThe sample size was not predetermined and simply results from the repetition of experiments. No sample that survived the experiment, as assessed by the continuation of cell divisions, was excluded. No randomization method was used. The investigators were not blinded during experiments.https://github.com/MechaBlasto/PeCoWaCo.git.The code used to analyze the oscillation frequencies from PIV and local curvature analyses can be found at WizardofOz can be found under the MTrack repository.The Fiji plug-in for local curvature analysis S1 FigAnalysis of PeCoWaCo during cleavage stages using PIV and Fourier transform. (A) Peak value divided by mean signal of the respective power spectra after Fourier analysis of PIV data for embryos at the zygote, 2-, 4-, and 8-cell stages. Embryos are classified as showing a visible oscillation (+) or not (−), as assessed visually. (B) ROC curve resulting from CutOffFinder [(C–F) Classification of zygote (C), 2- (D), 4- (E), and 8-cell stages (F) using a threshold of 1.777 for the peak/mean amplitude. Red indicates a mismatch between the visual assessment and the use of the threshold to determine whether a given embryo oscillates or not at a given time point. (G) Proportion of early and late 2-cell , 4-cell , and 8-cell stage  embryos showing detectable oscillations after Fourier transform of PIV analysis. Error bars show SEM. Chi-squared p-values comparing different stages are indicated ((H) Oscillation period of early and late 2-cell , 4-cell , and 8-cell  stages embryos. Larger circles show median values. Student t test p-values are indicated Click here for additional data file.S2 FigSurface tension of Vx-680 and fragmented embryos. (A) Surface tension of embryos treated with DMSO (n = 12) or Vx-680 (n = 13). Student t test p-value is indicated ((B) Surface tension of mechanical control (n = 14) or fragmented cells (n = 14). Student t test p-value is indicated (ndicated . Larger ndicated . Larger (PDF)Click here for additional data file.S3 FigSurface tension of fused blastomeres. (A) Surface tension of cells resulting from the fusion of 8, 4, or 2 16-cell stage blastomeres . Student t test p-value is indicated ((B) Radius of fused and fragmented 16-cell stage blastomeres. Larger circles show median values Click here for additional data file.S4 FigComparison of PIV and curvature analyses of PeCoWaCo. (A) Representative images of a 4-cell stage embryo overlaid with a subset of velocity vectors from PIV analysis (top left) or a color coded local curvature analysis (top right). Velocity over time for a representative velocity vector (bottom left) or local curvature measurement (bottom right) for the bottom most blastomere. White scale bar, 10 μm. Colored scale bar indicates curvature. See (B) Oscillation period of 4-cell stage embryos (n = 6) as determined by PIV or curvature tracking analyses. Pairwise Student t test p-value is indicated (ndicated . PeCoWaC(PDF)Click here for additional data file.S5 FigMolecular and structural characterization of the actomyosin cortex during cleavage stages. (A) Representative image of the actin cortex of a Phalloidin-stained 2-cell stage embryo acquired using super resolution microscopy. Scale bar, 5 μm. Overlaid white line shows the segmentation of the cortex, and orthogonal blue and yellow lines show line scans outside and inside the cell, respectively. Schematic diagram below shows example of where in the cell such image is taken. (B) Phalloidin intensity along lines orthogonal to the cell cortex of the cell shown in (A) . Mean intensity profile shown in red. (C) Cortex thickness measured at the zygote, 2-, 4-, and 8-cell stage . Student t test p-values are indicated ((D–G) mRNA (D and E) and protein (F and G) levels during cleavage stages for formins (D and F) and arps (E and G) adapted from [(H) Immunostaining of FMNL3 (gray and cyan in merged image) on representative zygote, 2-, 4-, and 8-cell stage embryos. Phalloidin shown in magenta on the merged image. Scale bar, 20 μm.ndicated , S1 Datated from . (H) Imm(PDF)Click here for additional data file.S6 FigMolecular and structural characterization of the actomyosin cortex during cleavage stages. (A) Schematic diagram of GFP-Fmnl3 mRNA injection in one blastomere of a 2-cell stage embryo. (B) Immunostaining of a 4-cell stage embryo injected with GFP-Fmnl3 at the 2-cell stage. GFP-Fmnl3 is shown on the left, and FMNL3 immunostaining is shown in gray in the middle and in cyan on the right together with Phalloidin in magenta. Scale bar, 20 μm. (C) Live imaging of a 4-cell stage embryo injected with GFP-Fmnl3 at the 2-cell stage. Left shows a merged image of phase contrast and GFP-Fmnl3. Right image shows the same embryo overlaid with a subset of velocity vectors from PIV analysis. Graphs on the right are velocity over time for a representative velocity vector of an uninjected (left) and GFP-Fmnl3 injected blastomere (right). Scale bar, 20 μm. See (D) Schematic diagram of GFP or GFP-Fmnl3 mRNA injection in both blastomeres of a 2-cell stage embryo. (E) Representative images of the preimplantation development of GFP or GFP-Fmnl3 expressing embryos shown at the 4-, 8-, 16-cell, and blastocyst stages. Scale bar, 20 μm. (PDF)Click here for additional data file.S1 Tablep-Values from chi-squared test for PeCoWaCo detection and from Student t test for period comparisons. Red when above 0.05, green when below 0.01, and black in between. See (DOCX)Click here for additional data file.S2 Tablep-Values from chi-squared test for PeCoWaCo detection and from Student t test for period comparisons. Red when above 0.05, green when below 0.01, and black in between. See (DOCX)Click here for additional data file.S3 Tablep-Values from chi-squared test for PeCoWaCo detection and from Student t test for period comparisons. Red when above 0.05, green when below 0.01, and black in between. See (DOCX)Click here for additional data file.S4 Tablep-Values from Student t test. Red when above 0.05, green when below 0.01, and black in between. See (DOCX)Click here for additional data file.S5 Tablep-Values from Student t test. Red when above 0.05, green when below 0.01, and black in between. See (DOCX)Click here for additional data file.S6 Tablep-Values from Student t test. Red when above 0.05, green when below 0.01, and black in between. See (DOCX)Click here for additional data file.S7 Tablep-Values from chi-squared test for PeCoWaCo detection and from Student t test for period and surface tension comparisons. Red when above 0.05, green when below 0.01, and black in between. See (DOCX)Click here for additional data file.S8 Tablep-Values from chi-squared test for PeCoWaCo detection and from Student t test for period and surface tension comparisons. Red when above 0.05, green when below 0.01, and black in between. See (DOCX)Click here for additional data file.S1 DataIndividual tabs are named according to the corresponding figure panels.(XLSX)Click here for additional data file.S1 MovieTime-lapse imaging of zygote, 2-, 4-, and 8-cell stage embryos showing PeCoWaCo. Pictures are taken every 5 seconds, and PIV analysis is performed between 2 consecutive images. PIV vectors are overlaid on top of the images with vectors pointing upward in magenta and downward in green. Scale bar, 20 μm. PeCoWaCo, periodic cortical waves of contraction; PIV, Particle Image Velocimetry.(AVI)Click here for additional data file.S2 MovieTime-lapse imaging of 4- to 8-cell stage embryos before, during and after undergoing their third cleavage. Pictures are taken every 5 seconds. Scale bar, 20 μm. PeCoWaCo, periodic cortical waves of contraction.(AVI)Click here for additional data file.S3 MovieTime-lapse imaging of 4-cell stage embryos showing PeCoWaCo after treatment with DMSO or 2.5 μM Vx680 at the time of the second cleavage division. Pictures are taken every 5 seconds, and PIV analysis is performed between 2 consecutive images. PIV vectors are overlaid on top of the images with vectors pointing upward in magenta and downward in green. Scale bar, 20 μm. PeCoWaCo, periodic cortical waves of contraction; PIV, Particle Image Velocimetry.(AVI)Click here for additional data file.S4 MovieTime-lapse imaging of mechanically manipulated and fragmented 2-cell stage blastomeres with and without nucleus. Pictures are taken every 5 seconds, and PIV analysis is performed between 2 consecutive images. PIV vectors are overlaid on top of the images with vectors pointing upward in magenta and downward in green. Scale bar, 20 μm. PIV, Particle Image Velocimetry.(AVI)Click here for additional data file.S5 MovieMontage of mTmG (top) and local curvature measurements (bottom) of fused 8×, 4×, 2× 1/16th blastomeres showing PeCoWaCo. Scale bar, 20 μm. PeCoWaCo, periodic cortical waves of contraction.(AVI)Click here for additional data file.S6 MovieMontage of mTmG (top) and local curvature measurements (bottom) of mechanically manipulated and fragmented 16-cell stage blastomeres with and without nucleus 1/16th blastomeres showing PeCoWaCo. Scale bar, 10 μm. PeCoWaCo, periodic cortical waves of contraction.(AVI)Click here for additional data file.S7 MovieTime-lapse imaging of a 4-cell stage embryos. PIV analysis is performed between 2 consecutive images. PIV vectors are overlaid on top of the images with vectors pointing upward in magenta and downward in green. Curvature analysis is performed by locally fitting a circle to the cell surface. Pictures are taken every 5 seconds. Scale bar, 20 μm. PIV, Particle Image Velocimetry.(AVI)Click here for additional data file.S8 MovieTime-lapse imaging of 2-cell stage embryos treated with DMSO or 100 nM Latrunculin A (LatA) showing PeCoWaCo. Pictures are taken every 5 seconds, and PIV analysis is performed between 2 consecutive images. PIV vectors are overlaid on top of the images with vectors pointing upward in magenta and downward in green. Scale bar, 20 μm. PeCoWaCo, periodic cortical waves of contraction; PIV, Particle Image Velocimetry.(AVI)Click here for additional data file.S9 MovieTime-lapse imaging of 4-cell stage embryos expressing GFP-Fmnl3 in 2 blastomeres. First frame shows the merge of GFP-Fmnl3 (green) and phase contrast (gray) images. Pictures are taken every 5 seconds, and PIV analysis is performed between 2 consecutive images. PIV vectors are overlaid on top of the images with vectors pointing upward in magenta and downward in green. Scale bar, 20 μm. PIV, Particle Image Velocimetry.(AVI)Click here for additional data file.S10 MovieTime-lapse imaging of 4-cell stage embryos expressing GFP or GFP-Fmnl3. Pictures are taken every 5 seconds, and PIV analysis is performed between 2 consecutive images. PIV vectors are overlaid on top of the images with vectors pointing upward in magenta and downward in green. Scale bar, 20 μm. PIV, Particle Image Velocimetry.(AVI)Click here for additional data file.S11 MovieTime-lapse imaging of 4-cell stage embryos expressing GFP or GFP-Fmnl3. Pictures of GFP (green) and phase contrast (gray) are taken every 3 hours. Scale bar, 20 μm.(AVI)Click here for additional data file. 23 Sep 2021Attachment118346_1_rebuttal_2180637_qzqbfm.docxSubmitted filename: Click here for additional data file. 6 Oct 2021Dear Dr Maitre, Thank you for submitting your manuscript entitled "Zygotic contractility awakening during mouse preimplantation development" for consideration as a Short Report by PLOS Biology.Your manuscript, reviews from Review Commons, and revision plan have now been evaluated by the PLOS Biology editorial staff as well as by an Academic Editor with relevant expertise and I am writing to let you know that we would like to consider a revised version of your manuscript that addresses the reviewer comments from Review Commons.Before we can invite you to submit a revised manuscript, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.Once you have completed your submission, we will send you a formal "major revision" decision, which will include additional comments from the Academic Editor and a 3 month deadline for the revision.Please re-submit your manuscript within two working days, i.e. by Oct 08 2021 11:59PM.https://www.editorialmanager.com/pbiologyLogin to Editorial Manager here: http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF when you re-submit. During resubmission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit Given the disruptions resulting from the ongoing COVID-19 pandemic, please expect delays in the editorial process. We apologize in advance for any inconvenience caused and will do our best to minimize impact as far as possible.plosbiology@plos.org if you have any queries relating to your submission.Feel free to email us at Kind regards,LucasLucas SmithAssociate EditorPLOS Biologylsmith@plos.org 8 Oct 2021Attachment118346_1_rebuttal_2180637_qzqbfm.docxSubmitted filename: Click here for additional data file. 11 Oct 2021Dear Dr Maitre,Thank you for submitting your manuscript "Zygotic contractility awakening during mouse preimplantation development" for consideration as a Short Report at PLOS Biology. As mentioned in our last email, your manuscript, the reviews from Review Commons, and your revision plan have been evaluated by the PLOS Biology editors and an Academic Editor with relevant expertise.In light of the reviews, which I have appended below, we will not be able to accept the current version of the manuscript. However, we would welcome re-submission of a much-revised version that takes into account the reviewers' comments as outlined in your submission. Having discussed your revision plan with the Academic Editor, we would like to emphasize the need to strengthen the evidence from the latrunculin experiments by a) providing measurements of changes in surface tension b) providing alternative methods to manipulate surface tension . We also would emphasize the need to measure surface tension in size-manipulated embryos.We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent for further evaluation by the reviewers. We expect to receive your revised manuscript within 3 months. plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.Please email us  and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/*PLOS Data Policy*http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information . For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5Please note that as a condition of publication PLOS' data policy  such that it can be cited independently in the future. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Lucas SmithAssociate EditorPLOS Biologylsmith@plos.org*****************************************************REVIEWS:Reviewer # 1Evidence, reproducibility and clarity:Periodic waves of contraction (PeCoWaCo) are stereotypical phenomena conserved in many oocytes and embryos that can lead to large-scale shape changes. However, their function is often unclear, and the parameters controlling their characteristics  still elusive in many model systems. In this manuscript, Ozguc and collaborators study the initiation of zygotic contractility during preimplantation development in the mouse. They show that PeCoWaCo become detectable in early embryos after the 2-cell stage, and progressively increase in frequency with each successive cleavage. Using elegant and challenging macro-manipulations of mouse embryos, they show that PeCoWaCo are not initiated by a reduction in cell size inherent in cell cleavage, nor their oscillation properties. Instead, PeCoWaCo are initiated and amplified by a gradual decrease in surface tension from the zygote to the 8-cell stage. The authors suggest that cortical softening awakens zygotic contractility.The question addressed by the authors is an important one, in several fields  and could therefore have wide impact. However, although the data presented are convincing, they fall short, controls are lacking to fully support the conclusions, and easy experiments could complement the existing limited results and take the paper to a higher level, making it a more complete study.**Major comments:**-Since the authors talk about "cortical maturation" during early embryogenesis and briefly about actin polymerization, they should describe the cortex itself during early embryogenesis . This could also help to understand why embryos decrease their surface tension during early development.-The authors use only one mean to reduce the tension of the cortex, using low doses of Latrunculin A. First, the authors should actually show that surface tension is reduced after this treatment by measuring it by micropipette aspiration, as they do in Figure 4 for control embryos, and show actin organization after such treatment (by immunofluorescence or live imaging). Second, could they use other means to reduce the tension of the cortex ? Third, could they do the reverse, increase the tension of the cortex after the 2-cell stage to test if this inhibits/decreases PeCoWaCo ? Finally, what is the long-term effect of disrupting the appearance/characteristics of PeCoWaCo on subsequent embryo development? This question could be addressed by tuning the intensity of PeCoWaCo using the experiments suggested above in the zygote or at the 2- cell stage, and monitoring embryo development up to the blastocyst stage to assess the outcome on embryo development.-In most figures, with the exception of Figure 3, the authors detect and analyze PeCoWaCo using PIV, a technique that tracks movements in the cytoplasm. However, cytoplasmic movements are not always due to the contractility of the cortex and PeCoWaCo. They can be generated by cortex-independent cytoplasmic activity. Thus, it would have been preferable to follow the cell contour as in Figure 3 for all figures, for all stages . Another way to validate PIV as a means of detecting PeCoWaCo without having to repeat all the experiments could be to correlate PIV with oocyte contour in control embryos at all stages.-In the treatments perturbing the cell size to test whether it is sufficient to initiate PeCoWaCo  or modify their periodicity , how is the surface tension between all conditions? Indeed, the authors nicely show that the cell size does not matter for PeCoWaCo, but that the cell surface does matter, so if PeCoWaCo are similar between cells of different sizes, it means that their surface tension should be in the same range as well. The authors should therefore measure it by micropipette aspiration.- re the characteristics of the PeCoWaCo the same in interphase and mitosis? In Bischof 2017, surface contraction waves in starfish oocytes are regulated by a cdk1-cyclinB gradient in meiosis. Could such a mechanism play a role in what the authors observe in early mouse embryos? For example, is there a correlation between cell cycle length  and PeCoWaCo strength? If so (shorter cell cycles/M-phases in zygotes compared to 8-cell stage embryos), one could imagine that mouse embryos are progressively exposed to cdk1-cyclinB for longer periods of time during their development, which would contribute to the appearance and increase in PeCoWaCo.**Minor comments:**-Why does the proportion of embryos with oscillations decrease at the 8-cell stage compared to the 4-cell stage ? It should not, since the surface tension is lower in 8-cell stage compared to the 4-cell stage .-Figure 2G shows that PeCoWaCo are not detected in either control or fragmented cells at the 2-cell stage but with a volume corresponding to a 4-cell stage, suggesting that 4-cell stage blastomere size is not sufficient to trigger PeCoWaCo. However, in Figure 1D, PeCoWaCo are detected in half of the control embryos at the 2-cell stage. Why is there a difference in the % of control embryos at the 2-cell stage with oscillations (0% versus 50%) between these 2 experiments? Could this be due to the CCD treatment allowing volume reduction  in Figure 2G?-Since the authors fuse and dissociate embryos at the 16-cell stage, they should show the period and velocity of PeCoWaCo in unmanipulated 16-cell stage embryos prior to any treatment.-In Figure 3, the percentage of embryos with detected oscillations should be added to the figure, as for Figure 1D or 2G.-In Figure 3C-F, the period of the PeCoWaCo is about 100 s, higher than that of the 8-cell stage in Fig1E, which is about 75 s. Is this difference statistically significant? And if so, why would it be higher in Figure 3?-Since PeCoWaCo become detectable after the 2nd cleavage, and thus after the ZGA, it could depend on a development program, allowing the surface tension to be reduced. The authors could have discussed this point further in the discussionFinally, the figures are difficult to read and the legends lack precision:-Some x-axis should be more informative with more than 3 values .- he value of the averages, or the exact percentages, should appear in full somewhere in the text, figures or legends .-The exact p-value should be written in full somewhere in the text, figures or legends .-Sometimes the titles of the axes do not correspond to what the graph shows. For example, in graphs 1D, 2C, 2G, 4D, S1A, the x-axis shows all the embryos, not just the ones with oscillations.-some figure legends lack information: What are the white arrows in Figure3A? What are we looking at in Figure 3A, embryos in transmitted light or expressing a probe to follow the embryo contour? T and V in Figure 3A represent period and velocity, but it is not stated in the legend. The curvature bar in Figure 3A is strange as it is mostly white. The scale bar values are missing for Figures 2B, 2F, 4A and 4C.-In Figure 3, the y-axes are not all consistent, sometimes the radius of the embryos is shown, sometimes fractions of 16-cell stage cells (which is not easy to read). SIGNIFICANCE: The question addressed by the authors is an important one, in several fields  and could therefore have wide impact. However, although the data presented are convincing, they fall short, controls are lacking to fully support the conclusions, and easy experiments could complement the existing limited results and take the paper to a higher level, making it a more complete study.Reviewer # 2Evidence, reproducibility and clarity:The manuscript by Özgüc and coworkers analyses the cortical contraction waves that are observed in early mouse embryos around the 8- to 16-cell stage. First, they provide a very careful, precise quantitative characterization of these contraction waves using PIV (particle image velocitometry) and by measuring the local cortex curvature. They find that contraction waves 'awaken' as development progresses, i.e. the oscillation frequency (and amplitude) of the waves is increasing during the cleavage divisions.Then, they use a number of very elegant micromanipulations to dissect whether the decreasing cell size is the cause for the increased cortical activity and increased frequency of contraction waves. Somewhat surprisingly, but very clearly they demonstrate that cortical contractions are independent of cell size. These results are consistent across a wide range of manipulated cell (fragment) sizes, and various types of treatments.Next, they measure the surface tension of cells using micropipette suction. These experiments reveal a clear correlation between surface tension and the period of contraction waves. To prove the causal relation between these processes, they artificially decrease the surface tension in 2-cell embryos by partially depolymerizing the cortex using Latrunculin A. This led to a strengthening of the contractions.Together, the authors conclude that properties of cortical contraction waves are independent of cell size. Instead, they propose that the reason for the gradually increasing frequency of cortical contraction waves is the gradual softening of the cell cortex accompanying cleavage divisions.**Major comments:**1.The authors start introducing contraction waves by quantifying cytoplasmic flows using PIV. I would find it important to first show that deformations of the cortex are indeed causing the observed cytoplasmic flow patterns, which should be straightforward to analyze by using the tools shown on Fig. 3A. As there are other known, cortex independent mechanisms to generate cytoplasmic flows, these experiments would be important to exclude such alternative mechanisms.2.I am very impressed by the various manipulations the authors use to demonstrate the independence of cell size and cortical contractions. However, I wonder why these manipulations have not been used in combination with surface tension measurements. Even more critically, I am missing more experiments that would directly demonstrate the causal relationship between cortical tension and contraction period. Unfortunately, the Latrunculin A results are less clear (as compared to other results shown in the paper), and the effect may indeed be more complex. For example, Bement et al  suggested a biochemical feedback mechanism based on F-actin, which in this experiment could not be distinguished from 'mechanical' effects of surface tension. Generally, I feel that additional perturbation experiments are needed in order to strengthen the conclusions. This should include upstream pathways known to regulate cortical contractility such as RhoA, for example.**Minor comment:**I would not mind if the authors used PeCoWaCo as an acronym through the present manuscript, but as they state, this is likely to be a very conserved feature of the cell cortex possibly of all animal cells. With this in mind, I doubtthat the community would adopt such a rather complicated acronym to describe this process.SIGNIFICANCE:Waves of cortical contractions are likely to be a widely conserved feature of the cortex of animal cells. The present manuscript makes important contributions to understand this process by (i) developing tools to quantify the process, (ii) show that contraction waves are independent of cell size, (iii) the period of contractions likely depend on the surface tension of the cell. These findings are broadly relevant for the cell biology community as a whole, however, as stated above, the findings needs to be substantially strengthened before publication, in my opinion. 10 Jan 2022Attachment220106ResponseReferees.docxSubmitted filename: Click here for additional data file. 7 Feb 2022Dear Dr Maitre,Thank you for submitting your revised Short Reports entitled "Zygotic contractility awakening during mouse preimplantation development" for publication in PLOS Biology. I have now obtained advice from the original reviewers and have discussed their comments with the Academic Editor. Based on the reviews (attached below), we will probably accept this manuscript for publication, provided you satisfactorily address the remaining points raised by the reviewers. Please also make sure to address the following data and other policy-related requests.Please also note that our Short Reports only have a maximum of 4 main figures, thus please redistribute the information currently shown in Figure 5 in the remaining figures or as a Supplementary Figure.In addition, we would like to consider a suggestion to improve the title:"Cortical softening mediated by Fmnl3 downregulation awakens zygotic contractility during mouse preimplantation development"Please only use 'awakens' if it is an actual term used in the field. Otherwise, "elicits" would probably be more appropriate.As you address these items, please take this last chance to review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the cover letter that accompanies your revised manuscript.We expect to receive your revised manuscript within two weeks. https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:To submit your revision, please go to -  a cover letter that should detail your responses to any editorial requests, if applicable, and whether changes have been made to the reference list-  a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable)-  a track-changes file indicating any changes that you have made to the manuscript. NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:https://journals.plos.org/plosbiology/s/supporting-information  *Published Peer Review History*Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/*Early Version*Please note that an uncorrected proof of your manuscript will be published online ahead of the final version, unless you opted out when submitting your manuscript. If, for any reason, you do not want an earlier version of your manuscript published online, uncheck the box. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.*Protocols deposition*https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsTo enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at Please do not hesitate to contact me should you have any questions.Sincerely,Ines--Ines Alvarez-Garcia, PhD,Senior Editor,ialvarez-garcia@plos.org,PLOS Biology------------------------------------------------------------------------DATA POLICY: IMPORTANT - PLEASE READhttp://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797 You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:1) Supplementary files . Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to  using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication. Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it:Fig. 1B-E; Fig. 2C, D, G, F; Fig. 3C, D, F, G, I-L; Fig. 4C, E-G; Fig. 5B-D; Fig. S1A-H; Fig. S2A-B; Fig. S3A-B; Fig. S4A-B; Fig. S5B-G and Fig. S6CNOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived .Please also ensure that figure legends in your manuscript include information on WHERE THE UNDERLYING DATA CAN BE FOUND, and ensure your supplemental data file/s has a legend.Additionally, please provide all the accession numbers and make the files publicly available at this stage.Please ensure that your Data Statement in the submission system accurately describes where your data can be found.------------------------------------------------------------------Reviewers' commentsRev. 1:As stated in my previous review for Review Commons, the manuscript has three major conclusions: (i) it provides a quantitative description of oscillatory cortical contraction waves in early mouse embryos; (ii) it shows using a series of perturbation and micromanipulation experiments that frequency and amplitude of these contraction waves is independent of cell size; (iii) it provides correlative evidence and a couple of perturbation experiments suggesting that progressive softening of the oocyte cortex is the cause of the observed 'awakening' of contractions as cleavage divisions proceed.My major criticism was the lack of direct evidence for a causal relationship between cortical tension and contraction waves. This criticism was partially addressed by the authors in the revised manuscript by combining the perturbation experiment (low-dose latrunculin treatment) with measurement of the cortical tension, and by including an additional perturbation (overexpression of fmnl3). With these additions, I think the manuscript is very well suited for publication in PLoS Biology as a Short Report, as I consider the findings still somewhat preliminary, but certainly of high relevance, which is the exact purpose of the Short Reports, as I understand.I only have a few minor comments to improve readability:1. As I explained in my previous review, I do think it is very important to establish that PIV is a valid approach to report on cortical contractions. Therefore, I would suggest moving the panels now shown on FS4 into ideally Figure 1 or into one of the other main figures.2. I would suggest to include the data on FS6 in the last main figure. I think this is a very a nice experiment!3. I would suggest removing FS5 panel H, because without detailed validation these stainings are not easily interpretable.Rev. 2:The authors responded to all my comments by arguing or adding experiments, and the result is that the article is strengthened .One key question remains: what is the physiological role of these contractions ? In other word, what is the long-term effect of disrupting the appearance/characteristics of PeCoWaCo on the subsequent development of the embryo? This question could be addressed by tuning the intensity of PeCoWaCo in the zygote or at the 2-cell stage, and monitoring the development of the embryo up to the blastocyst stage to assess the outcome on embryo development. This question was only partly addressed by Fmnl3 overexpression, as the effect is transient: theembryos show PeCoWaCo at the end of the 4-cell stage and develop to blastocyst normally. However, since this manuscript is considered a Short Report, this is not necessary and will pave the way for future research.Minor:- The scale bar values are still missing in the legends for Figures 2B, 2F, 4A, 4D 5A.- The tables legends are wrong sometimes . 18 Feb 2022Attachment220215ResponseToReferees.docxSubmitted filename: Click here for additional data file. 4 Mar 2022Dear Jean-Leon,On behalf of my colleagues and the Academic Editor, Sally Lowell, I am pleased to say that we can in principle accept your Short Report entitled "Cortical softening elicits zygotic contractility during mouse preimplantation development" for publication in PLOS Biology, provided you address any remaining formatting and reporting issues. These will be detailed in an email that will follow this letter and that you will usually receive within 2-3 business days, during which time no action is required from you. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have any requested changes.http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.Please take a minute to log into Editorial Manager at PRESSbiologypress@plos.org. If you have previously opted in to the early version process, we ask that you notify us immediately of any press plans so that we may opt out on your behalf.We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with http://www.plos.org/about/media-inquiries/embargo-policy/.We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. Sincerely, Ines--Ines Alvarez-Garcia, PhD Senior Editor PLOS Biologyialvarez-garcia@plos.org


In the present study, we confirmed that α-asaronol, which is a product of the active metabolites of alpha Asarone, did not affect n-butylphthalide efficacy when n-butylphthalide and α-asaronol were co-administered to rats with cerebral ischemia-reperfusion injury. Our research revealed that the co-administration of α-asaronol and n-butylphthalide could further improve neurological function, reduce brain infarct volume, increase the number of Nissl bodies, and decrease the ratios of apoptotic cells and the expression of the caspase-3 protein for cerebral ischemia-reperfusion injury model compared to n-butylphthalide alone. Additionally, α-asaronol could significantly decrease the incidence of post-stroke epilepsy versus n-butylphthalide. This study provides valuable data for the follow-up prodrug research of α-asaronol and n-butylphthalide. Stroke, a nervous system disease which is caused by acute cerebral blood circulation disorder, is the second leading global cause of death and a leading cause of long-term disability . It posePatients with ischemic strokes are susceptible to a variety of complications, of which epilepsy can further lead to the aggravation of hypoxia/anoxia in brain tissue, ultimately adversely affecting patients’ recovery of motor function . This coIn recent decades, Chinese herbal medicines, which have thousands of years of history of use in therapy, have become increasingly popular in clinical use due to their good safety profiles and few toxic side effects ,16. Alphp < 0.05, CA-HD p < 0.001, NBP p < 0.01). The difference in the scores of mNSS of CA-LD and CA-HD versus NBP was not statistically significant. Further, the score obtained for CA-HD performed slightly better than that for NBP. These results show that ASOL did not affect NBP efficacy when NBP and ASOL were co-administered. On the other hand, high-dose α-asaronol (CA-HD) was beneficial to the recovery of the neurological function of rats with cerebral ischemia-reperfusion injury.In the present study, in order to explore the effect of ASOL in the presence of NBP on cerebral ischemia-reperfusion injury, we investigated a model of cerebral ischemia-reperfusion injury. The vehicle group had the highest score of mNSS among the five groups, which revealed that the modeling was successful A. Comparp < 0.01, CA-HD p < 0.001, NBP p < 0.01). Furthermore, compared with NBP, there was no statistical significance for CA-LD and CA-HD. Additionally, the infarct volume of CA-HD performed slightly betterthan that for NBP. This further confirmed the suggestion made above that a high dose of α-asaronol (CA-HD) was beneficial for the recovery of rats with cerebral ischemia-reperfusion injury.The un-infarcted area was red and the infarcted area was white in the representative photographs of TTC staining for rat with cerebral ischemia-reperfusion injury B. The rep < 0.01). Furthermore, compared with NBP, CA-HD showed a very significant reduction in the incidence of post-stroke epilepsy (p < 0.05). It was suggested that ASOL treatment may be an effective means to prevent the occurrence and development of post-stroke epilepsy in the presence of NBP for rats with cerebral ischemia-reperfusion injury.Post-stroke epilepsy is a very common complication in stroke survivors ; as suchPost-stroke epilepsy, which accounts for almost 50% of newly diagnosed epilepsy in patients with an age greater than 60 years, could lead to the aggravation of the disease state, seriously affect the prognosis, and even endanger life . Howeverp < 0.001). Both CA-HD and NBP showed remarkable increases in the number of Nissl bodies compared with the vehicle group . Furthermore, the number of Nissl bodies in CA-HD was shown to be slightly higher than that in NBP. This is consistent with the above findings.Nissl bodies are a marker of mature neurons that reflect the functional state of neurons and can be used to detect neuronal apoptosis . In thisTUNEL staining is a method that is used to detect the DNA fragmentation generated during apoptosis . TUNEL-pp < 0.01). After the administration of CA-HD and NBP, the expression of the caspase-3 protein significantly decreased  compared to that in the vehicle group. There was no significant difference between the CA-HD and NBP groups, but the CA-HD group showed a lower mean optical density than the NBP’s for the expression of caspase-3. These results further confirm that α-asaronol was beneficial to the recovery of rats with cerebral ischemia-reperfusion injury.The activation and expression of caspase-3, a cysteine protease, preceded neuronal damage, which ultimately leads to cell apoptosis ,29,30. TIn this study, we firstly confirmed that α-asaronol did not affect the efficacy of NBP when NBP and α-asaronol were co-administered to rats with cerebral ischemia-reperfusion injury. Subsequently, we determined that the co-administration of α-asaronol and NBP could further improve neurological function, reduce brain infarct volume, increase the number of Nissl bodies, and decrease the ratios of apoptotic cells and the expression of the caspase-3 protein compared to NBP alone. More importantly, α-asaronol could significantly decrease the incidence of post-stroke epilepsy versus NBP. These positive results substantiate the notion that α-asaronol treatment is an effective means to prevent the occurrence and development of post-stroke epilepsy. A prodrug could be made via an esterification reaction from hydroxyl groups of α-asaronol and carboxylic groups which the ester hydrolysis of NBP afforded.The results of this study provide a theoretical basis for follow-up prodrug research on α-asaronol and NBP.α-asaronol (purity: >99%) was kindly provided by Prof. Hu from Chongqing Medical University. Racemic 3-n-butylphthalide was purchased from Macklin. ASOL and NBP were dissolved in 5–10% Solutol HS-15 and 90–95% saline.Male Sprague Dawley rats (200~220 g) were obtained from the Animal Laboratory Center of Chongqing Medical University . All animals were housed under a 12 h light–dark cycle ad libitum in a temperature-controlled (25 ± 1 °C) facility and maintained on standard food and water. All animal experimental protocols were in accordance with the guidelines of the National Institutes of Health and approved by the Experimental Ethics Committee of Chongqing Medical University (license number: SYXK (YU) 2010-001).All animals were fasted for 10–12 h before the study but had free access to water. All animal experiments were performed in a blinded setup. The MCAO model was established according to the literature with minor modifications . Brieflyn = 12): sham, vehicle, coadministration of NBP (10 mg/kg) and ASOL (15 mg/kg) (CA-LD), coadministration of NBP (10 mg/kg) and ASOL (30 mg/kg) (CA-HD), and administration of NBP (10 mg/kg) only. After 24 h of cerebral ischemia-reperfusion, the neurological deficit scores were assessed blindly according to the modified neurological severity score (mNSS) system [Sixty male adult SD rats (200~230 g) were randomly distributed into five groups  system . The mNSThe brain tissue of each group was quickly isolated, frozen for 30 min at −20 °C, and cut into 2 mm-thick coronal sections after a neurological function evaluation. The sections were then stained with 2% 2,3,5-triphenyltetrazolium chloride  in phosphate-buffered saline (PBS) for 30 min in the dark and fixed with 4% paraformaldehyde (PFA) for 24 h. These slices stained with TTC were photographed and the infarction areas were measured using the Image J 1.52p analysis software . The calculation of the percentage of infarct volume was performed with the sum infarct area of the ipsilateral hemisphere/the sum area of the hemisphere.n = 22): sham, vehicle, coadministration of NBP (10 mg/kg) and ASOL (15 mg/kg) (CA-LD), coadministration of NBP (10 mg/kg) and ASOL (30 mg/kg) (CA-HD), and administration of NBP (10 mg/kg) only. Rats were closely observed for the occurrence of tonic-clonic seizures and status epilepticus in the 24 h after cerebral ischemia-reperfusion and administration.SD rats (200~230 g) were randomly divided into five groups  to closely pack the brain tissues to the slides. After the coronal sections were dewaxed and rehydrated, these sections were stained with a toluidine blue solution, washed with distilled water, soaked in 70% alcohol, and differentiated in 95% alcohol. Sections were then subjected to dehydration with anhydrous ethanol, cleaned with xylene until transparent, and sealed with neutral gum. Nissl staining images were acquired from the peri-infarct cortex area with a Panoramic 250 digital slice scanner  and analyzed via the Image J 1.52p analysis software . The mean density of neurons was calculated from up to three non-overlapping and representative images obtained from the brain sections of rats from different groups.TUNEL staining was performed according to the manufacturer’s instructions using the cell death detection kit . In brief, after being deparaffinized and rehydrated, the sections were repaired with the citrate repair solution (pH 6.0) and washed with phosphate-buffered saline (PBS). TUNEL staining solution was added and the sections were incubated in dark for 60 min at room temperature. After rinsing, the sections were then stained with DAPI . Finally, the sections were washed with PBS and sealed with a glycerin gelatin mixture. TUNEL staining images were acquired from the peri-infarct cortex area with a Panoramic 250 digital slice scanner  and analyzed via the Image J analysis software . The TUNEL-positive cells and the total number of nuclei were counted from up to three non-overlapping and representative images obtained from the brain sections of rats from different groups, and the ratios of apoptotic cells were then calculated in each group.2O2 in methanol for 10 min, then washed with PBS for 5 min at RT; this process was repeated three times. Subsequently, the sections were dipped into citrate solution (pH 6.0), heated to boiling, and then cooled at room temperature. After 10 min, the heating and cooling cycle was repeated once. After being cooled to room temperature, sections were washed with PBS twice for 6 min each at room temperature. The sections were blocked with normal goat serum  at RT for 25 min and incubated with primary antibody against caspase-3  at 4 °C for 12 h. Then, sections were incubated with the secondary antibody of biotin-labeled goat anti-rabbit  at 37 °C for 40 min and washed with PBS three times for 6 min each time. The slides were developed with the DAB chromogen kit , their color development was checked under a microscope, and then they were washed with distilled water. Finally, the sections were slightly counterstained with hematoxylin, dehydrated, transparentized, and sealed with neutral gum. Sections were scanned using a Panoramic 250 Digital Slide Scanner  and analyzed using Image-Pro Plus 6.0 . Integrated optical density (IOD) and the positive area of all images in the peri-infarct cortical neurons were measured from up to three non-overlapping and representative images obtained from the brain sections of rats from the different groups. The calculation of the mean optical density (MOD) of each image was performed by dividing the IOD by the positive area.After dewaxing sections, the sections were treated with 3% Hp < 0.05 were considered statistically significant.All data were analyzed using the IBM SPSS Statistics 26.0 software , and the graphical representations were performed via GraphPad Prism 8.0 software . If the data followed a normal distribution and the groups had equal variances, multiple comparisons among groups were analyzed via a one-way analysis of variance (ANOVA), followed by LSD post hoc analysis. If the data followed a normal distribution and the groups had unequal variances, multiple comparisons among groups were evaluated by a one-way ANOVA with Dunnett’s T3 test. The incidence of post-stroke epilepsy was analyzed using the chi-square test for comparisons among groups. Values of


The fight against the current coronavirus disease 2019 (COVID-19) pandemic has created a huge demand of biotechnological, pharmaceutical, research and sanitary materials at unprecedented scales. One of the most urgent demands affects the diagnostic tests. The growing need for rapid and accurate laboratory diagnostic tests requires the development of biotechnological processes aimed at producing reagents able to cope with this demand in a scalable, cost-effective manner, with rapid turnaround times. This is particularly applicable to the antigens employed in serological tests. Recombinant protein expression using plants as biofactories is particularly suitable for mass production of protein antigens useful in serological diagnosis, with a neat advantage in economic terms.Nicotiana benthamiana plants. After purification, the recombinant N protein obtained was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to SARS-CoV-2 in human sera. To validate the ELISA, a panel of 416 sera from exposed personnel at essential services in Madrid City Council were tested, and the results compared to those obtained by another ELISA, already validated, used as reference. Furthermore, a subset of samples for which RT-PCR results were available were used to confirm sensitivity and specificity of the test.We expressed a large portion of the nucleoprotein (N) derived from SARS-CoV-2 in The performance of the N protein expressed in plants as antigen in serologic test for SARS-CoV-2 antibody detection was shown to be highly satisfactory, with calculated diagnostic sensitivity of 96.41% (95% CI: 93.05–98.44) and diagnostic specificity of 96.37 (95% CI: 93.05–98.44) as compared to the reference ELISA, with a kappa (K) value of 0.928 (95% CI:0.892–0.964). Furthermore, the ELISA developed with plant-derived N antigen detected SARS-CoV-2 antibodies in 84 out of 93 sera from individuals showing RT-PCR positive results (86/93 for the reference ELISA).This study demonstrates that the N protein part derived from SARS-CoV-2 expressed in plants performs as a perfectly valid antigen for use in COVID-19 diagnosis. Furthermore, our results support the use of this plant platform for expression of recombinant proteins as reagents for COVID-19 diagnosis. This platform stands out as a convenient and advantageous production system, fit-for-purpose to cope with the current demand of this type of biologicals in a cost-effective manner, making diagnostic kits more affordable. The present SARS-CoV-2 coronavirus pandemic is the most important global sanitary crisis humankind has faced in the last 100 years. Since its emergence in Asia at the end of 2019, the new virus has already hit all continents, producing harm of remarkable magnitude both in public health, and in the World economy. The pandemic has also underscored the need of rapid and flexible responses in the industrial capacities required to produce large amounts of tools and reagents for diagnosis, therapies, and prevention (vaccines) of new viral diseases in a cost-effective way.Serological tests are essential tools for the detection of antibody responses to SARS-CoV-2 infection. They play an essential role not only in diagnosis but also in the evaluation of the seroprevalence in exposed populations and in research studies aimed to provide answers to still open key questions regarding the complete characterization of the course of the infection such as, for instance, the duration of antibodies. In this regard, enzyme-linked immunosorbent assays (ELISAs) are widely used in most diagnostic and research applications due to their long-proved efficiency, easiness, and high throughput capacity.Many serological assays have been made available recently for detection of SARS-CoV-2 specific antibodies . HoweverNicotiana benthamiana. The favorable features of this industrial approach have been widely and frequently reviewed, among which speed of production in very large amounts and comparative low costs of productions stand out  and shown its suitability to detect antibodies using a wide panel of human serum samples obtained in early 2020. For that, we developed an ELISA test which was used to analyze the presence of antibodies to SARS-CoV2 N antigen in the panel sera. The results were compared with those obtained using an already validated ELISA test commercially available.Many serological assays for SARS-CoV-2 antibody detection rely upon the use of the viral multifunctional nucleoprotein (N) , expressN. benthamiana plants. The sequence was cloned into TMV expression vector (GENEWARE System) under the control of a duplicated promoter of the capsid protein (CP) gene. The extensin signal peptide (PSN) from Nicotiana tabacum, which directs the recombinant NPC-2 toward the plant endoplasmic reticulum  of SARSCoV-2 which codes for its amino acids 231–419 (GenBank YP_009724397.2) was designed with a His-tag at its C-terminus. The synthetic sequence was made by GENEART AG optimizing for codon usage in eticulum , was alsPlasmid DNA of the stable clone TMV-PSN-NPC-2 was purified using QIAGEN’s plasmid purification kit, according to the manufacturer’s instructions.in vitro from plasmid TMV-PSN NPC-2 clone with the mMESSAGE mMACHINETM T7 Transcription Kit (INVITROGEN). Each in vitro transcription reaction (IVT) was made with 0.625 μl plasmid DNA (0.2 μg/μl), 0.125 μl water, 0.25 μl 10× Buffer, 1.25 μl 10× nucleotides (CAP) and 0.25 μl 10× T7 RNA polymerase, in a 2.5 μl final volume. The reaction was incubated for 2 h at 37°C. RNA was quantified by electrophoresis in a 0.5% ethidium bromide-stained agarose gel ]. Plants were kept at 25°C in growth chambers .Three hundred N. benthamiana plants in a greenhouse . Infection and NPC-2 expression were assessed by ELISA with polyclonal antibodies against TMV  and SARSCoV-2 N protein  according to the manufacturers’ instructions.Infected leaves from 5 dpi symptomatic plants were pooled and extracted by grinding in 150 ml of 50 mM phosphate buffer, pH 7.5, with 0.04% (w/v) of sodium metabisulphite. The crude extract was then used to inoculate 2,000 Stability of the recombinant virus during infection was evaluated by Immunocapture Reverse Transcription Polymerase Chain Reaction (IC-RT-PCR) according to g to remove cell debris and insoluble material. The soluble extract was subjected to a two-step purification process involving a first stage by Ni2+-sepharose affinity chromatography, and a second stage by Cation Exchange in a HiTrap SP Sepharose FF 5 ml (AKTA Xpress System -GE), according to Symptomatic leaves were harvested 11–13 dpi and blender-ground in 100 mM potassium phosphate buffer, pH 7.3–7.6, 100 mM NaCl, 0.04% (v/v) Triton ×100, 5 mM BME, and 0.1 mM PMSF (1:3 w/v). The homogenate was filtered through three cloth layers and centrifuged at 6,000 × The purified recombinant NPC-2 protein was subjected to SDS-PAGE gel analysis (100–500 ng/lane) and stained with Coomassie Blue. The bands with a migration in the gel around 21 kDa, were excised. Their tryptic fragments were analyzed by MALDI-TOF in an external service .The purified NPC-2 was subjected to Western blot analysis, probed with a commercial polyclonal antibody specific for SARSCoV-2 N protein . For that, recombinant NPC-2 protein (0.3–3.0 μg) was separated by SDS-PAGE and electrotransferred into a nitrocellulose membrane, which was blocked with TBS buffer supplemented with 5% (w/v) skimmed milk, and subsequently incubated with the commercial anti-SARSCoV-2 N polyclonal antibody, diluted 1:2,000, for 1 h. The development was done after incubation with alkaline-phosphatase-conjugated goat anti-rabbit IgG (97048 Abcam), 1:200, 1 h, followed by substrate addition (NBT/BCIP).n = 193) or negative (n = 223) to SARS-CoV-2 antibodies on the basis of their result with a commercially available ELISA . already validated, with 100% diagnostic sensitivity  and 98.2% diagnostic specificity (95% CI = 97–99.1%)  . A subse7–99.1%)  in oroph7–99.1%) .2SO4. Optical density (OD) was measured in each well at 450 nm in an ELISA microplate reader. Results were valid when OD in negative control wells was below 0.8 OD units and the OD in positive control wells was over 2.50 OD units.Enzyme-linked immunosorbent assay plates  were coated with 2 μg/well of purified recombinant NPC-2 protein antigen in 0.1 M carbonate/bicarbonate buffer pH 9.6 and maintained at 37°C for 45 min. Thereafter, wells were blocked with phosphate buffered saline, pH 7.4, 0.05% (v/v) Tween 20 (PBST), containing 3% (p/v) skimmed milk, for 1 h at room temperature (RT). The plates were incubated with serum samples diluted 1:200 in PBST with 1% skimmed milk, for 2 h at RT. Duplicates of positive (sera from a confirmed COVID 19 patient), and negative controls were included in each plate. Plates were washed three times with PBST and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Thermofisher), diluted 1:5,000 in PBST, for 1 h at RT. Finally, after three additional washes with PBST, plates were incubated with peroxidase substrate  for 5 min in the dark at RT. Reaction was stopped by adding 0.5 M HData were statistically analyzed using a receiver-operator characteristics (ROC) curve analysis using the GraphPad Prism 6 software. The kappa statistic, analyzed by the same software, was used to measure the strength of agreement between the results obtained by the indirect ELISA test with recombinant NPC-2 protein antigen and the reference commercial ELISA.Sensitivity of the tests (NPC-2 ELISA and reference ELISA) was calculated as the proportion of positive samples by the method used as reference (reference ELISA or samples from patients with previous positive PCR result) that tested also as positive in the evaluated test.Specificity was calculated as the proportion of negative samples by reference ELISA which tested negative in NPC3 ELISA.N. benthamiana plants was successfully cloned into TMV-PSN vector and the recombinant TMV-PSN-NCP2 clone was confirmed by sequencing. In vitro 6 kb RNA transcripts produced from the recombinant TMV-PSN-NCP2 clone were used to infect plants by mechanical inoculation , designed with His-tag C-terminal and optimized to achieve its correct expression in culation . Evidentculation .N. benthamiana plants by IC-RT-PCR at 8 and 12 dpi. Amplification of an 820-bp fragment, which is the size expected for the recombinant clone, was obtained in 100% of the plants analyzed with TMV-PSN-NPC-2 while in the different controls included, namely, extracts from plants infected with the empty vector (TMV-PSN) and from plants infected with another unrelated construct (TMV-PSN EGF) the amplicons observed were of 226 and 397 bp, respectively, corresponding with their expected sizes  but also that 1 in 8 plants, both in 8 and 12 dpi, showed a partial deletion of the insert (600 bp).2+-affinity and cationic exchange. After Ni2+-affinity chromatography, more than 90% of the NPC-2 protein was recovered from the soluble plant extract, with over 80% purity  of SARS CoV-2 N protein (data not shown).N. benthamiana plants . The ELISA was optimized for IgG antibody detection in serum samples (data not shown). A total of 416 human serum samples were analyzed, that, in the absence of a serological gold standard method, were previously classified as positive or negative on the basis of the results obtained with a commercial ELISA (hereinafter “reference ELISA”) that has proven to be highly sensitive and specific , useful as antigen for serologic tests, in plants of  respect . Each on respect .Escherichia coli . Just to give an idea of the potential of this production system, one ELISA plate using the format described in this study requires 0.2 mg antigen, so 12 g may yield enough antigen to fill 60,000 plates and analyze approximately 5,400,000 serum samples. According to our own calculations the approach followed in this study might reduce the production costs of serologic tests significantly, making them more affordable, especially in low-income countries. Only two purification steps were required to obtain >95% purity: A first initial affinity chromatography capture, followed by a second stage of polishing through cationic exchange. After the two processes, over 90% of the protein was recovered. The recombinant protein was identified by MALDI-TOF and proven to be recognized by a commercial antibody raised against the full viral protein. No sign of protein degradation was observed in the purification process. One of the main drawbacks of viral vectors for protein production in plants is the stability of the foreign gene in the plant cell during their replication cycle. This particular issue was addressed by IC-RT-PCR carried out on infected plants 8–12 dpi, that is, the period coinciding with tissue harvest. We found a remarkably good stability with 12.5% of the plants showing partial deletions. Under our growth conditions, plants at 12 dpi were reaching a weight of 1.2–1.8 g/leaf.In this study, the C-terminal domain of the N protein (aa 231–419) has been expressed in proteins , 2014. Tproteins , and alsproteins . An initThe ability of plant-made recombinant N protein to be recognized by antibodies was evaluated in 416 serum samples from infected and non-infected individuals collected during the first epidemic wave  affecting Spain in the context of a surveillance study focused on voluntarily participating active staff from the essential services of Madrid City Council . This panel of samples was reflecting the immunological status of this cohort of staff occupationally exposed to virus infection in a context of high virus transmission. Moreover, RT-PCR from nasal swabs was performed in each individual at the time of blood sample collection and, in some cases, also earlier, in order to verify their infection status. Thus, it was possible for a subset of serum samples used in this study to ensure they came from ongoing infections. It is widely known that there is a lag period between the detection of SARS-CoV-2 RNA in nasopharyngeal tract and the development of antibodies in serum. Thus, it has been observed that some RT-PCR positive individuals yield negative results in serologic assays . This maDifferential IgG levels could be detected by ELISA in sera from COVID-19 patients with a similar or a better sensitivity, as compared to other systems using recombinant N protein, as the reference ELISA used in this work. The assay showed a high sensitivity in patients recently infected so it can represent an interesting approach for diagnostic and epidemiological studies. The high purity, sensitivity in antibody detection, and very low level of endotoxins in the final protein preparation should make it a powerful diagnostic tool. From an industrial standpoint, the yields obtained in the purification process would allow to manufacture recombinant protein for millions of diagnostic reactions kits in 8 weeks .Very recently, another study has been published showing the usefulness of other plant-produced SARS-CoV-2-derived antigens, the S (or “spike”) protein and its Receptor-Binding Domain (RBD) . Apart fAlthough not specifically tested in this study, cross-reaction with other coronaviruses currently circulating in humans  is not expected due to the low percentages of identity observed in the amino acid sequence of homologous regions of our truncated version of SARS-CoV-2 nucleoprotein (NPC-2) in the human coronaviruses, such as MERS (51%), OC43 (37%), HKU1 (36%), NL63 (29%), and 229E (27%).Upon validation of the results and technology presented in this work, we should be in a position to tackle the production of other SARS-CoV-2 proteins. This would allow increased specificity in the detection of virus variants.Finally, it is worth to remark that in this study, technology derived from the first virus ever described  is used to produce a protein from the last human described virus  at an industrial scale, and enables the development of cost-efficient serological tests useful to fight its infection, and INGENASA for providing the ELISA kits used as reference technique.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.PL, LW, FP, and MJ-C conceived this article. LW, SJ, and FL designed the experiments. LW and PL drafted the first manuscript with support from FP. SJ, AR, SG, AS, IB, FL, and LW carried out the experiments. MM-C and BP-G contributed to sample management and procurement. LW and FL analyzed data and performed statistical analyses. All authors discussed the results and contributed to the final manuscript.LW, SJ, AR, SG, AS, IB, and PL were employed by the company Agrenvec S.L. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.